Three isoforms of NM II: NM II-A, II-B and II-C are expressed in mammals. Ablation of NM II-A in mice (A-/A-) leads to embryonic lethality by E6.5 with disorganized germ layer formation. Mice ablated for NM II-B (B-/B-) die by E14.5 with defects in heart and brain development. The cardiac defects include a decrease in the number of cardiac myocytes which can be attributed in part to abnormalities in cytokinesis. To understand the function of NM II-C in vivo, we generated mice ablated for NM II-C (C-/C-). The C-/C- mice are viable and show no obvious abnormalities during development, but show mild phenotypic changes in the heart, including a decrease in cardiac function as measured by MRI at 6 months and moderate myocyte hypertrophy at one year. The presence of mild cardiac abnormalities in C-/C- mice prompted us to cross these mice with II-B hypomorphic mice which express only 12% of the wild type levels of NM II-B in the heart. The homozygous offspring of this mating (BIC-/BIC-) develop cardiac myocyte hypertrophy as well as interstitial fibrosis at 6 months, a time when both C-/C- and BI/BI mice show no such abnormalities. To further examine this effect in cardiac myocytes, we generated II-B and II-C double knockout mice (B-C-/B-C-). Similar to B-/B- mice, B-C-/B-C- mice died by E14.5 with a hypoplastic heart. In addition to a failure in cytokinesis, B-C-/B-C- cardiac myocytes also showed major defects in karyokinesis. During mitosis many B-C-/B-C- myocytes demonstrate defects in chromatid separation and spindle formation. While defective karyokinesis in the cardiac myocytes was occasionally observed (15%) in B-/B- mice, the frequency was increased to 90% in B-C-/B-C- mice. The requirement for NM II in karyokinesis was further demonstrated in cultured HL-1 atrial myocytes using siRNA knockdown of NM II or blebbistatin. Unlike the cardiac myocytes where NM II-B is the predominant isoform and II-A is not detected, HL-1 cells express 95% NM II-A and 5% II-B. An additive effect on karyokinesis in HL-1 cells was seen following siRNA knockdown of NM II-A and II-B. In summary we present evidence that NM II isoforms play a role in cardiac myocytes cytokinesis and karyokinesis.